Hughson A G, Lee G F, Hazelbauer G L
Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4660, USA.
Protein Sci. 1997 Feb;6(2):315-22. doi: 10.1002/pro.5560060206.
Oxidative crosslinking of cysteines introduced by site-specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)-(o-phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining transmembrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1') in the other subunit and a more distant placement of TM2 and TM2', the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1-TM1' interaction and TM2-TM2' orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.
通过定点诱变引入的半胱氨酸的氧化交联是蛋白质结构分析的有力工具，但该方法仅限于体外研究。我们最近报道，大肠杆菌的完整细胞可以用Cu(II)-(邻菲罗啉)3或分子碘处理，这样不会干扰鞭毛功能或一般趋化反应，然而在化学感受器Trg相邻跨膜螺旋中的特定半胱氨酸之间会定量形成交联。这表明氧化交联可用于体内结构分析。因此，我们使用了我们全面收集的Trg衍生物，每个衍生物在受体单体两个跨膜段的54个位置之一含有一个半胱氨酸，以表征这种同二聚体蛋白在体内和体外的交联模式。我们发现，作为一种结构分析技术，体内交联与更传统的体外方法相比具有优势。完整细胞氧化处理产生的交联模式表明跨膜段1 (TM1)与其在另一个亚基中的同源伙伴(TM1')广泛相互作用，以及TM2和TM2'的位置更远，这与在分离膜中交联确定的关系相同。此外，在体内和体外都确定了TM1-TM1'相互作用和TM2-TM2'取向的相同螺旋面。模式的对应也表明，通过体外交联分析确定的结构特征与化学感受器在其天然环境即完整、有功能的细胞中的组织有关。看来用于体内氧化的两种功能良性处理的不同特征可以提供对蛋白质动力学的见解。
